title: Defined culture medium for stem cell differentiation: applicability of serum-free conditions in the mouse embryonic stem cell test.
abstract: The embryonic stem cell test (EST) is a validated method to assess the developmental toxicity potency of chemicals. It was developed to reduce animal use and allow faster testing for hazard assessment. The cells used in this method are maintained and differentiated in media containing foetal calf serum. This animal product is of considerable variation in quality, and individual batches require extensive testing for their applicability in the EST. Moreover, its production involves a large number of foetuses and possible animal suffering. We demonstrate the serum-free medium and feeder cell-free maintenance of the mouse embryonic stem cell line D3 and investigate the use of specific growth factors for induction of cardiac differentiation. Using a combination of bone morphogenetic protein-2, bone morphogenetic protein-4, activin A and ascorbic acid, embryoid bodies efficiently differentiated into contracting myocardium. Additionally, examining levels of intracellular marker proteins by flow cytometry not only confirmed differentiation into cardiomyocytes, but demonstrated significant differentiation into neuronal cells in the same time frame. Thus, this approach might allow for simultaneous detection of developmental effects on both early mesodermal and neuroectodermal differentiation. The serum-free conditions for maintenance and differentiation of D3 cells described here enhance the transferability and standardisation and hence the performance of the EST.
authors.affiliation: German Federal Institute for Risk Assessment (BfR), Centre for Alternative Methods to Animal Experiments-ZEBET, 12277 Berlin, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Riebeling
authors.forename: Katharina
authors.initials: K
authors.lastname: Schlechter
authors.forename: Roland
authors.initials: R
authors.lastname: Buesen
authors.forename: Horst
authors.initials: H
authors.lastname: Spielmann
authors.forename: Andreas
authors.initials: A
authors.lastname: Luch
authors.forename: Andrea
authors.initials: A
authors.lastname: Seiler
mesh_terms: D000826:Animal Testing Alternatives; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects*; D002478:Cells, Cultured; D003470:Culture Media; D053595:Embryonic Stem Cells / Q000187:drug effects*; D005434:Flow Cytometry; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology; D051379:Mice; D009200:Myocardial Contraction; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000378:metabolism
chemical_list: D015415:Biomarkers; D003470:Culture Media; D036341:Intercellular Signaling Peptides and Proteins